Project Summary/Abstract In the United States, cancers account for 1 out of every 4 fatalities, and remain the second most common cause of death. Since the 1940s, natural products (NPs) or derivatives thereof have been critical in the treatment of these diseases, amounting to 74% of cancer drugs on the market. Unfortunately, NP drug discovery has received relatively small investment in recent decades, resulting in downsizing or complete elimination of such programs in the pharmaceutical industry. This is partly due to inefficiencies related to bioactivity-based screening of complex NP mixtures in the traditional discovery paradigm, including (1) limited or unreliable sources for source material, (2) the inability of high-throughput screening to identify interesting NPs of low abundance within complex extracts and (3) the high probability of known compound reisolation. Interestingly, because only 1% of bacteria can be cultivated in the laboratory, and because cultured organisms only produce a small subset of their biosynthetic potential, it has become abundantly clear that microorganisms have the genetic capacity to produce a far greater number of NPs than have been isolated. Thus, methods for exploiting this abundant, virtually untapped biological source of new compounds are desperately needed. Recent efforts in large-scale profiling of bacterial metabolomes have identified large numbers of potentially novel compounds, but how are these vast collections of data to be prioritized? The ?metabologenomics? platform enables organization of such data for specific targeting of novel antiproliferative compounds with simultaneous identification of their biosynthetic genes. The proposal herein details plans for mining of an entire bacterial library for a terminal reductase-containing subset of expressed NP gene clusters due to their favorability for anti-proliferative activity. In Aim 1, two confirmed novel compounds will be targeted for characterization based on antiproliferative-endowing properties evident by reductase domains present in their biosynthetic genes. Their antiproliferative capacity will be evaluated following structure elucidation and confirmation of biosynthesis. This targeted approach will ensure that resources are directed toward compounds with the greatest likelihood for useful anticancer activity. The use of molecular networking in Aim 2 will add to the pair of molecules identified in Aim 1 by identifying analogs for isolation from across our bacterial library. This approach, inspired by structure activity relationship (SAR) studies in medicinal chemistry, will harness the biosynthetic potential of our entire library for the diversification and bioactivity-based prioritization of this novel class. This modern platform aims to accelerate discovery novel drug-leads for the clinical treatment of cancer and will generate new small-molecule probes for fundamental investigations into cancer biology.